In the past, immunological binding partners such as antibodies and fragments thereof have been used to specifically target molecular sites in vivo. When these immunological binding partners are attached to other chemical moieties, such as radionuclides, such chemical moieties can be delivered specifically to the target sites. Some degree of success has been achieved in specifically localizing radioactivity to tumor markers in vivo utilizing radiolabeled immunological binding partners. Specifically localized radioactivity has been used in vivo for both diagnostic purposes and therapy. For example, radiolabeled immunological binding partners have been used to diagnose deep venous thrombi, to study lymph node pathology, and to detect, stage, and treat neoplasms. Although polyclonal antibodies have previously shown promise for localizing to neoplasms, the development of monoclonal antibodies has provided even greater selectivity of binding and thus more specific targeting of the antibody in vivo.
One of the problems that has accompanied the use of immunological binding partners for diagnosis and therapy has been nonspecific, nontargeted delivery to undesirable sites, even with monoclonal antibodies. For example, administration of radiolabeled antibodies in vivo can result in an undesirable level of background radiation to nontargeted sites such as the liver. Significant background activity can remain for several days after injection even though radiolabeled intact antibody is cleared relatively rapidly from the bloodstream. One approach to reducing the nonspecific delivery of antibodies in vivo has been to fragment the antibodies and to utilize only the portion of the antibodies that specifically binds to an antigen.
To facilitate further discussion of antibody fragments, the following is a brief review of the structure of antibodies. In general, it is well known that antibodies are bifunctional molecules made up of four chains of amino acids and a variety of domains. A simplified model for an IgG antibody molecule showing the basic four-chain structure and domains is shown in FIG. 1. V indicates the variable regions, C the constant regions, and the vertical arrow indicates the so-called hinge region. Thick lines represent heavy (H) and light (L) chains. The thin lines between chains represent disulfide bonds. Cleavage by the enzymes papain and pepsin, which cleave at points indicated in FIG. 1, separates the so-called crystallizable fragment (Fc) from the antigen binding fragment (Fab) region of the antibody. More particularly, cleavage by papain results in two monovalent Fab fragments, whereas cleavage by pepsin produces a single divalent F(ab').sub.2 fragment held together by one or more disulfide bonds between the heavy chains.
It has been previously recognized that the use of Fab and F(ab').sub.2 fragments in radiolabeled form for therapy or diagnosis in vivo can result in reduced radioactive background in vivo, at least in part due to faster clearance rates of these fragments from serum as compared to the intact antibody. Moreover, Fc mediated liver uptake and macrophage binding are also eliminated by use of the Fab or F(ab').sub.2 fragments. Other advantages of the use of these antibody fragments as compared to intact antibody are the following: more rapid tissue distribution, reduced immunogenicity, and enhanced permeability across membranes.
Of the two types of fragments, Fab and F(ab').sub.2, the latter have been determined to have an ideal serum half-life. In general, if the serum half-life is too long, greater amounts of nonspecific targeting will occur. On the other hand, if the serum half-life is too short, not enough specific localization will occur. The serum half-life of murine F(ab').sub.2 fragments in a human is intermediate (about 7 to 8 hours) between intact antibody (24 hours or more) and Fab fragments (1 to 2 hours) and hence, is an advantageous median value. This advantage of the use of F(ab').sub.2 fragments has led to the prediction that these fragments will assume an increasingly important role in immunodiagnostic and therapeutic systems in vivo. See The Journal of Nuclear Medicine, 24(4):316-325 (1983).
Examples of studies in which F(ab').sub.2 fragments have been utilized, in radiolabeled form, are the following: The Journal of Nuclear Medicine, 27:685-693(1986); The Journal of Clinical Investigation, 77:301-311 (1986); and Cancer Research, 45:3378-3387 (1985).
In spite of the advantages described above for F(ab').sub.2 antibody fragments in the context of specific delivery of diagnostic and therapeutic agents such as radionuclides to target sites in vivo, some technical problems have arisen in producing such fragments. For example, the present inventors have attempted to make F(ab').sub.2 fragments from an IgG2b antibody referred to as NR-ML-05. Upon cleavage of the intact antibody with pepsin, no useful F(ab').sub.2 fragments are produced; rather, monovalent fragments were produced. See Handbook of Experimental Immunology, Vol. 1:Immunochemistry, Weir, 4th ed., Blackwell Scientific Pub. (1986). Apparently, the F(ab').sub.2 fragments are not stable to the cleavage conditions. For other antibodies, although the F(ab').sub.2 fragments may be made, attempts to attach them to other chemical moieties has resulted in reduction of specific binding by the antibody or cleavage to the corresponding Fab' fragment.
Accordingly, in spite of the previous advances in this field, there has remained a need for methods of producing stabilized antibody fragments, especially F(ab').sub.2 fragments, so that they may be used more widely.